Blocking endothelial protein C receptor (EPCR) accelerates thrombus development in vivo

Summary

The endothelial protein C receptor (EPCR) plays an anticoagulant role by improving protein C activation. Although low levels of activated protein C (APC) constitute a thrombosis risk factor, the relationship between modulating EPCR function and thrombosis has not been addressed so far. Monoclonal antibodies (mAb) against murine EPCR were raised, and their ability to block protein C/APC binding was tested. The ferric chloride carotid artery injury model in mice was chosen to test the effect of anti-EPCR mAb on thrombus formation. The time to total occlusion of the vessel was analysed in three groups, given an isotype control mAb (IC), a blocking (RCR-16) or a non-blocking (RCR-20) anti-EPCR mAb. RCR-16 prevented the interaction between protein C/APC and EPCR as demonstrated by surface plasmon resonance and flow cytometry, and inhibited the activation of protein C on the endothelium. IC and RCR-20 were unable to induce such effects. In vivo, RCR-16 shortened the time to total vessel occlusion with respect to IC [13.4 ± 1.0 (mean ± SD) and 17.8 ± 3.2 minutes, respectively, p<0.001]. Occlusive thrombi lasting for more than one hour were observed in all RCR-16-treated animals, but only in 43% of IC-treated ones. Results with RCR-20 were indistinguishable from those observed with IC. For the first time, a direct relationship between blocking EPCR and thrombosis is demonstrated. Blocking anti-EPCR autoantibodies can predispose to thrombosis episodes and may constitute a new therapeutic target.

Keywords

Endothelial cell protein C receptor - protein C - thrombosis - monoclonal antibodies - animal thrombosis models

• References

• 1 Fukudome K, Esmon CT. Identification, cloning, and regulation of a novel endothelial cell protein C/activated protein C receptor. J Biol Chem 1994; 269: 26486-26491.
• 2 Stearns-Kurosawa DJ, Kurosawa S, Mollica JS. et al. The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex. Proc Natl Acad Sci USA 1996; 93: 10212-10216.
• 3 Taylor Jr FB, Peer GT, Lockhart MS. et al. Endothelial cell protein C receptor plays an important role in protein C activation in vivo. Blood 2001; 97: 1685-1688.
• 4 España F, Vayá A, Mira Y. et al. Low level of circulating activated protein C is a risk factor for venous thromboembolism. Thromb Haemost 2001; 86: 1368-1373.
• 5 Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109: 3161-3172.
• 6 López-Sagaseta J, Montes R, Puy C. et al. Binding of factor VIIa to the endothelial cell protein C receptor reduces its coagulant activity. J Thromb Haemost 2007; 05: 1817-1824.
• 7 Ghosh S, Pendurthi UR, Steinoe A. et al. Endothelial cell protein C receptor acts as a cellular receptor for factor VIIa on endothelium. J Biol Chem 2007; 282: 11849-11857.
• 8 Preston RJ, Ajzner E, Razzari C. et al. Multifunctional specificity of the protein C/activated protein C Gla domain. J Biol Chem 2006; 281: 28850-28857.
• 9 Hurtado V, Montes R, Gris JC. et al. Autoantibodies against EPCR are frequently found in antiphospholipid syndrome and are a risk factor for foetal death. Blood 2004; 104: 1369-1374.
• 10 Lavigne-Lissalde G, Cochery-Nouvellon E, Granier G. et al. Diffuse skin necrosis in a patient with an anti-endothelial cell protein C receptor autoantibody which blocks protein C activation. J Thromb Haemost 2005; 03: 413-415.
• 11 Castellino FJ, Liang Z, Volkir SP. et al. Mice with a severe deficiency of the endothelial protein C receptor gene develop, survive, and reproduce normally, and do not present with enhanced arterial thrombosis after challenge. Thromb Haemost 2002; 88: 462-472.
• 12 Taylor Jr FB, Stearns-Kurosawa DJ, Kurosawa S. et al. The endothelial cell protein C receptor aids in host defense against Escherichia coli sepsis. Blood 2000; 95: 1680-1686.
• 13 Li W, Zheng X, Gu J. et al. Overexpressing endothelial cell protein C receptor alters the hemostatic balance and protects mice from endotoxin. J Thromb Haemost 2005; 03: 1351-1359.
• 14 Ye X, Fukudome K, Tsuneyoshi N. et al. The endothelial cell protein C receptor (EPCR) functions as a primary receptor for protein C activation on endothelial cells in arteries, veins, and capillaries. Biochem Biophys Res Commun 1999; 259: 671-677.
• 15 López-Sagaseta J, Montes R, Hermida J. Recombinant expression of biologically active murine soluble EPCR. Protein Expr Purif 2009; 64: 194-197.
• 16 Dahlbäck B, Villoutreix BO. Regulation of blood coagulation by the protein C anticoagulant pathway: novel insights into structure-function relationships and molecular recognition. Arterioscler Thromb Vasc Biol 2005; 25: 1311-1320.
• 17 Dahlbäck B. Advances in understanding pathogenic mechanisms of thrombophilic disorders. Blood 2008; 112: 19-27.
• 18 Oganesyan V, Oganesyan N, Terzyan S. et al. The crystal structure of the endothelial protein C receptor and a bound phospholipid. J Biol Chem 2002; 277: 24851-24854.
• 19 Van de Wouwer M, Collen D, Conway EM. Thrombomodulin-protein C-EPCR system: integrated to regulate coagulation and inflammation. Arterioscler Thromb Vasc Biol 2004; 24: 1374-1383.